Effects of chlorpyrifos and chlorpyrifos-oxon on the dynamics and movement of mitochondria in rat cortical neurons.

Program in Clinical and Experimental Therapeutics, University of Georgia, College of Pharmacy, Augusta, Georgia 30912-2450, USA.

Abstract

Organophosphate (OP)-based pesticides have been used extensively for decades, and as a result, they have become almost ubiquitous in our environment. There is clinical and animal evidence to suggest that chronic exposures to OPs can lead to cognitive dysfunction and other neurological abnormalities, although the mechanism for these effects is unknown. We previously reported that repeated, subthreshold exposures (defined as doses not associated with signs of acute toxicity) to the commonly used OP chlorpyrifos (CPF) resulted in protracted impairments in the performance of attention and memory-related tasks in rodents as well as deficits in axonal transport ex vivo (in the sciatic nerve). Here, we investigated the effects of CPF and its active metabolite CPF oxon (CPO) on the dynamics and movement of mitochondria in rat primary cortical neurons using time-lapse imaging techniques. Exposure to CPF (1.0-20.0 μM) or CPO (5.0 nM-20.0 μM) for 1 or 24 h resulted in a concentration-dependent increase in mitochondrial length, a decrease in mitochondrial number (indicative of increased fusion events), and a decrease in their movement in axons. The changes occurred at concentrations of CPF and CPO that did not inhibit acetylcholinesterase activity (the commonly cited mechanism of acute OP toxicity), and they were not blocked by cholinergic receptor antagonists. Furthermore, the changes did not seem to be associated with direct (OP-related) effects on mitochondrial viability or function (i.e., mitochondrial membrane potential or ATP production). The results suggest that an underlying mechanism of organophosphate-based deficits in cognitive function might involve alterations in mitochondrial dynamics and/or their transport in axons.

CPF disrupts mitochondrial transport and alters mitochondrial dynamics in cortical neurons. Representative images of cultures exposed to vehicle or 1.0 μM CPF for 24 h are provided in A and B, respectively. Scale bar, 100 μm. CPF exposure for 24 h was associated with a dose-dependent decrease in axonal transport (mean number of mitochondria moving per micrometer) (C), an increase in mitochondrial length (mean mitochondrial length within the region of interest) (D), a decrease in mitochondrial number (mean number of mitochondria per micrometer) (E). CPF exposure for 1 h was also associated with a dose-dependent decrease in mitochondrial movement (F), an increase in mitochondrial length (G), and a decrease in mitochondrial number (H). Data are expressed as the percentage of control ± S.E.M. *, significantly different (p < 0.05) from control.

CPO disrupts mitochondrial transport and alters mitochondrial dynamics in cortical neurons. Representative images of cultures exposed to vehicle or 0.005 μM CPO for 24 h are provided in A and B, respectively. Scale bar, 100 μm. CPO exposure of 24 h was associated with a decrease in axonal transport (mean number of mitochondria moving per micrometer) (C), an increase in mitochondrial length (mean mitochondrial length within the region of interest) (D), and a nearly significant (dose effect p < 0.054) decrease in mitochondrial number (mean number of mitochondria per micrometer) (E). CPO exposure for 1 h was also associated with a decrease in mitochondrial movement (F), an increase in mitochondrial length (G); and a decrease in mitochondrial number (H). Data are presented as the percentage of control ± S.E.M. *, significantly different (p < 0.05) from control; $, p < 0.09.

The nicotinic acetylcholine receptor antagonist mecamylamine does not block the effects of CPF or CPO on mitochondrial transport or dynamics. Various concentrations of mecamylamine were coincubated with 1.0 μM CPF or 0.005 μM CPO for 24 h. The results show that the axonal transport deficits (A and D) and increased mitochondrial length (B and E) induced by CPF and CPO, respectively, persisted in the presence of mecamylamine. The nonsignificant decreases in mitochondrial number associated with CPF (C) were also not antagonized, whereas the effects on the CPO-related effect on mitochondrial number (F) were less clear. Mecamylamine alone was not associated with significant effects on mitochondrial transport or length (A and B, right); however, it was associated with significant increases in number (C, right). Data are presented as mean (% control) ± S.E.M. *, significantly different (p < 0.05) from control; +, p < 0.09 versus control.

The muscarinic acetylcholine receptor antagonist atropine does not block the effects of CPF or CPO on mitochondrial transport or dynamics. Various concentrations of atropine were coincubated with 1.0 μM CPF or 0.005 μM CPO for 24 h. The results show that the axonal transport deficits (A and D) and increased mitochondrial length (B and E) induced by CPF and CPO, respectively, persisted (and increased even further) in the presence of atropine. The nonsignificant decreases in mitochondrial number associated with CPF and CPO (C and F, respectively) were also not antagonized by atropine. Atropine alone was not associated with significant effects on mitochondrial transport (A, right); however, it was associated with significant decreases in length and increases in number (B and C, right). Data are presented as mean (% control) ± S.E.M. *, significantly different (p < 0.05) from control.

Concentrations of CPF and CPO that alter mitochondrial transport and dynamics do not compromise ΔΨm. Cortical neurons were exposed to various concentrations of CPF or CPO for 24 h and analyzed via the DePsipher assay. A and B show representative images of vehicle and 20 μM CPF exposures, respectively. D and E show representative images of vehicle and 20 μM exposures to CPO, respectively. C and F show representative images of 500 μM exposures to CPF and CPO, respectively. G and H show the dose-effect relationships for CPF and CPO, respectively. Green images on the left (emission peak 527 nm) indicate the monomeric form of the DePsipher reagent in the cytoplasm of the neuron. Red images on the right (emission peak 590 nm) indicate the accumulation and aggregation of the reagent in healthy mitochondria (i.e., with an intact ΔΨm). Thus, 20 μM CPF and CPO were not associated with a compromise in ΔΨm, whereas the lack of red labeling in the cells exposed to 500 μM CPF or CPO indicates a compromise of the mitochondrial ΔΨm. For quantitative comparisons, the ratio of green to red fluorescence was assessed and OP-related effects were expressed as a percentage of the control ratios (i.e., as mean % control ± S.E.M.). *, significantly different (p < 0.05) from control.

Exposure to CPF and CPO does not significantly alter ATP synthesis or increase superoxide levels. Cortical neurons were incubated with various concentrations of CPF or CPO up to 20 μM or the positive control compound valinomycin (VAL), 5 μM for 24 h. ATP production was determined with a bioluminescent somatic cell assay kit (FLASC; Invitrogen). Exposure to CPF (A) or CPO (B), respectively, did not impair ATP production. ATP production was impaired by valinomycin. In subsequent experiments, cortical neurons were incubated with various concentrations of CPF or CPO up to 20 μM or the positive control compound antimycin A (Ant) and 5 or 50 μM for 24 h. Superoxide production was subsequently determined with MitoSOX Red kits. Exposure to CPF (C) or CPO (D), respectively, did not elevate superoxide levels. Superoxide levels were significantly (p < 0.05) elevated by the higher concentration of antimycin A. Data are presented as mean (% control) ± S.E.M. *, significantly different (p < 0.05) from control.